Mycoplasma pneumoniae is the causative agent of a primary atypical pneumonia, as well as milder respiratory tract infections, in humans. The ability to adhere to the respiratory epithelium is an important mycoplasma virulence determinant and appears to be a complex process requiring the proper interaction between several specific groups of mycoplasma proteins. Non-adhering variants arise spontaneously at a high frequency. Associated with the loss of adherence is the concomitant loss of either of the groups of adherence-associated proteins. This proposal focuses upon one such group of four proteins (designated HMW 1-4), and has as its goal the evaluation of the organization and regulation of the genes for HMW 1-4 in wild type and non-adhering variants of M. pneumoniae. Mycoplasma DNA will be cosmid-cloned into Escherichia coli, and clones will be screened for expression of the adherence-associated proteins using specific antibody probes for radioimmunopreciptation analysis. The cloned DNA will be characterized by restriction endonuclease mapping, DNA:DNA hybridization, and S1 nuclease mapping. Appropriate portions of the cloned genes will be evaluated for promoter regions using promoter-probe vectors. Gene organization will be determined by chromosome walking. DNA fragments with potential promoter activity will be sequenced. Comparison of gene organization and regulation in wild type and non-adhering variants will provide valuable information concerning the control of this important virulence factor.